Recently, the DNA sequence of human cancers has been investigated extensively and shown to contain more than 100,000 alterations in each cancer cell. These results have provided strong evidence to support our long-held hypothesis that normal mutation rates are insufficient to account for the multiple mutations present in cancer cells and that cancers exhibit a mutator phenotype. We have established a new method for next- generation DNA sequencing that gives us unprecedented accuracy. By sequencing both strands of individual DNA molecules we can detect a single mutation when present among 10 million wild type sequences. We desire to apply this to studying subclonal mutations in colon cancer, i.e. mutations that are present in only a small percent of the malignant cells within the tumor and would not be detected by routine next generation DNA sequencing. These mutations could be responsible for the ability of these cancers to circumvent host cell anti-cancer mechanisms and for the rapid emergence of resistance to chemotherapeutic agents in some cancers. We will carry out the following studies: 1) We will use our new method of double-stranded DNA sequencing to quantify the number of subclonal mutations as a function of the ability of individual cancers to proliferate, invade and metastasize; 2) We will determine if cance cell fitness is altered by specific types of mutations resulting from decreased fidelity of DNA replication or efficiency of DNA repair; and 3) We will use this ultra-accurate methodology to determine whether subclonal DNA mutations can be used as prognostic biomarkers. An understanding of the extent and sources of mutational heterogeneity within tumors may substantially affect our assessment of the disease, and greatly advance the delivery of comprehensive personalized chemotherapy.